Stability Kinetics of Imiquimod: Development and Validation of an Analytical Method.

For a new immune modulator imiquimod, various liquid chromatography methods have been described in literature but all of them are deficient in one or other aspects of complete method development. The present work intends to develop and validate the stability indicating reverse phase high performance liquid chromatographic (HPLC) method. The isocratic flow of mobile phase comprising equal volume ratio of acetate buffer BP pH 3.7 and acetonitrile at the rate of 1.5 mL/min through the C-18 column at 25°C lead to elution of drug around 2.3 min when analyzed at 244 nm using UV-detector. The linear regression equation in calibration plot was y = 61632×-1224 with 0.9992 coefficient of determination (r2). The percent relative standard variation (% RSD) in peak area at low, mid and high region of linearity range was less than 5% in precision studies. The method was able to detect 0.039 µg/mL of drug but practical limit of quantitation (LOQ) was 1.5 µg/mL. The imiquimod molecule was stable in all except oxidizing conditions where it degraded into more polar molecule in hydrogen peroxide (H2O2) concentration dependent manner. Therefore, an analytical method capable of accurately and specifically estimating the drug in microgram range was successfully developed.

Development and Validation of Stability Indicating UPLC-PDA/MS for the Determination of Imiquimod and its Eight Related Substances: Application to Topical Cream.

A stability indicating analytical method for imiquimod and its related impurities was developed by ultra-pressure liquid chromatography (UPLC) using design of experiments. This method could quantify imiquimod and all its eight known related impurities in a single run. The optimum separation was achieved on reverse phase Acquity UPLC column (2.1 mm × 100 mm, 1.7 µm) using 0.1% trifluoroacetic acid and acetonitrile as mobile phase. Preventing the use of ion pair reagents assured the compatibility of this method to liquid chromatography in tandem with mass spectrometry. All components were separated within 9 minutes, maintaining good resolution. The stability indicating nature of the developed method was assessed by analyzing the samples of imiquimod which were exposed to various environments such as acid, alkali, peroxide, light and heat. This method was found to be sensitive, precise and accurate. The method achieved the lower detection limit of 0.04 µg/mL and the quantification limit of 0.08 µg/mL for all analytes.